Human ubiquitin-conjugating enzyme homologs

ABSTRACT

The invention provides human ubiquitin-conjugating enzyme homologs (UCEH) and polynucleotides which identify and encode UCEH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of UCEH.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/058,368 filed on Apr. 9, 1998, entitled HUMANUBIQUITIN-CONJUGATING ENZYME HOMOLOGS, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof human ubiquitin-conjugating enzyme homologs and to the use of thesesequences in the diagnosis, treatment, and prevention of cancer,autoimmune disorders, and neuronal disorders.

BACKGROUND OF THE INVENTION

[0003] The ubiquitin system is a major pathway for selective proteindegradation. (Finley D. et al. (1991) Annu. Rev. Cell Biol. 7: 25-69.)Degradation by this system is instrumental in a variety of cellularfunctions such as DNA repair, cell cycle progression, signaltransduction, transcription, and antigen presentation. The ubiquitinpathway also eliminates proteins that are misfolded, misplaced, or thatare in other ways abnormal. This pathway requires the covalentattachment of ubiquitin (E1), a highly conserved 76 amino acid protein,to defined lysine residues of substrate proteins.

[0004] Substrate recognition by this pathway involves a specializedrecognition and targeting apparatus, known as the ubiquitin-conjugatingsystem. Ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase(E3), either independently or in conjunction, catalyze isopeptideformation between the carboxyl terminus of ubiquitin and amino groups ofinternal lysine residues of target proteins. (Scheffner M. et al. (1995)Nature 373: 81-83.) Ubiquitin-protein conjugates are then recognized anddegraded by a specific protease complex, the 26S proteasome. Both E2 andE3 exist as protein families, and their pattern of expression is thoughtto determine substrate specificity. (Nuber U. et al. (1996) J. Biol.Chem. 271: 2795-2800.) For example, E6 oncoprotein of thecancer-associated human papillomavirus types 16 and 18, inactivates thetumor suppressor protein p53 via the ubiquitin protein degradationpathway. An E3 protein, E6-AP, and an E2 protein, either UbcH5 or UbcH7,complex with E6 and specifically conjugate ubiquitin to p53. (ScheffnerM. et al. (1993) Cell 75: 495-505; Nuber et al., supra.) Other E2proteins are not sufficient for p53 ubiquitination, thus UbcH5 and UbcH7appear to be involved in the specific targeting of p53 for degradation.

[0005] The yeast ubiquitin-conjugating enzyme, Ubc3, also known asCDC34, plays a crucial role in the progression of the cell cycle fromthe G1 to S. Correct positioning of ubiquitin on a surface of Ubc3 is arequirement for cell cycle transition. (Prendergast J.A. et al. (1995)J. Biol. Chem. 270: 9347-9352.) Mutation studies have suggested thatamino acid residues S73, S97, and S139 of Ubc3 may be critical forsubstrate specificity, while C95 is the site of catalytic activity. (LiuY. et al. (1995) Mol. Cell Biol. 15: 5635-5644.) An alteration in C95and another highly conserved amino acid, L99, results in a dominantnegative mutation. (Banerjee A. et al. (1995) J. Biol. Chem. 270:26209-26215. ) Overexpression of this mutation of Ubc3 blocks cellgrowth in otherwise wild type strains.

[0006] A decrease in muscle mass, known as muscle wasting or cachexia,has been shown to be associated with the ubiquitin-dependent proteolyticsystem. Rats bearing the Yoshida AH-130 ascites hepatoma for 7 daysshowed a significant decrease in muscle mass in relation to non-tumorbearing controls. (Llovera M. et al. (1995) Int. J. Cancer 61: 138-141.) The muscle wasting was found to be associated with an increasedproteolytic rate related to the ubiquitin-dependent proteolytic system.Muscle wasting is common among human cancer patients. In addition tocancer, ubiquitin-dependent muscle wasting is also influenced bynutritional manipulation such as fasting and dietary protein deficiency,muscle activity and disuse, AIDS, and the pathological conditions,sepsis, trauma, and acidosis. (Attaix D. et al. (1994) Reprod. Nutr.Dev. 34: 583-597. ) In a rat model for long lasting sepsis, researchersfound that E2 mRNA levels increase during the acute and chronic diseasephases and parallel a rise in muscle protein breakdown. (Voisin L. etal. (1996) J. Clin. Invest. 97: 1610-1617.)

[0007] Evidence from experiments on mouse and rabbit reticulocytesindicates that ubiquitin conjugation is a key rate-limiting step inantigen presentation. (Grant E.P. et al. (1995) J. Immunol. 155:3750-3758.) The rates of degradation of beta-galactosidase constructscorrelated with the rates of class I antigen presentation in vivo. Thisshows that ubiquitin degradation pathways may have a critical role ingenerating major histocompatibility complex (MHC) class I-presentedpeptides.

[0008] The presence of ubiquitin and ubiquitin conjugates has beendetected in patients affected by neurodegenerative diseases such asAlzheimer's disease. Whereas the intracellular amyloid beta-proteinprecursor (APP) did not show appreciable ubiquitin-mediated degradation,three extracellular APP forms were degraded by this proteolytic pathway,suggesting a potential regulatory role for the ubiquitin-dependentsystem in the in vivo APP metabolic pathway. (Gregori L. et al. (1994)Biochem. Biophys. Res. Commun. 203: 1731-1738. ) Paired helicalfilaments (PHF) are fibrillar structures that accumulate in degeneratingneurons in the brains of Alzheimer's disease patients. One component ofPHF, the PHF-smear, consists of the tau protein fragment bound toubiquitin. (Morishima M. et al. (1994) Dementia 5: 282-288.)

[0009] Depletion of specific cellular proteins may have many medical andagricultural benefits. Redirecting the ubiquitin-dependent proteolyticpathway may facilitate specific proteolytic removal. Five examples inwhich target recognition by E2s was redefined by engineering E2s tocontain appropriate protein-binding peptides fused to their C terminihave been reported. (Gosink M. M. et al. (1995) Proc. Natl. Acad. Sci.92: 9117-9121.) Thus, it may be possible to design E2s capable ofdirecting the selective removal of many intracellular proteins, such asthose implicated in pathogenesis, for example in Alzheimer's disease.The ability to selectively modulate E2 activity may be useful fortreating diseases associated with protein degradation, such asAlzheimer's disease, muscle wasting syndrome, or for targeting undesiredproteins for degradation, such as in the case of viral infections andcancer.

[0010] The discovery of new human ubiquitin-conjugating enzyme homologsand the polynucleotides encoding them satisfies a need in the art byproviding new compositions which are useful in the diagnosis, treatment,and prevention of cancer, autoimmune disorders, and neuronal disorders.

SUMMARY OF THE INVENTION

[0011] The invention features substantially purified polypeptides, humanubiquitin-conjugating enzymes, referred to collectively as “UCEH” andindividually as “UCEH-1, ” “UCEH-2,” and “UCEH-3.” In one aspect, theinvention provides a substantially purified polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment ofSEQ ID NO: 2, and a fragment of SEQ ID NO: 3.

[0012] The invention further provides a substantially purified varianthaving at least 90% amino acid identity to the amino acid sequences ofSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or to a fragment of any ofthese sequences. The invention also provides an isolated and purifiedpolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO: 2,and a fragment of SEQ ID NO: 3. The invention also includes an isolatedand purified polynucleotide variant having at least 90% polynucleotidesequence identity to the polynucleotide encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, afragment of SEQ ID NO: 2, and a fragment of SEQ ID NO: 3.

[0013] Additionally, the invention provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO: 2,and a fragment of SEQ ID NO: 3, as well as an isolated and purifiedpolynucleotide having a sequence which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO: 2,and a fragment of SEQ ID NO: 3.

[0014] The invention also provides an isolated and purifiedpolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, a fragmentof SEQ ID NO: 4, a fragment of SEQ ID NO: 5, and a fragment of SEQ IDNO: 6. The invention further provides an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide sequenceidentity to the polynucleotide sequence comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, a fragment of SEQ ID NO: 4, a fragment of SEQ ID NO: 5,and a fragment of SEQ ID NO: 6, as well as an isolated and purifiedpolynucleotide having a sequence which is complementary to thepolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, a fragmentof SEQ ID NO: 4, a fragment of SEQ ID NO: 5, and a fragment of SEQ IDNO: 6.

[0015] The invention further provides an expression vector containing atleast a fragment of the polynucleotide encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, afragment of SEQ ID NO: 2, and a fragment of SEQ ID NO: 3. In anotheraspect, the expression vector is contained within a host cell.

[0016] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, afragment of SEQ ID NO: 2, and a fragment of SEQ ID NO: 3, the methodcomprising the steps of: (a) culturing the host cell containing anexpression vector containing at least a fragment of a polynucleotideencoding the polypeptide under conditions suitable for the expression ofthe polypeptide; and (b) recovering the polypeptide from the host cellculture.

[0017] The invention also provides a pharmaceutical compositioncomprising a substantially purified polypeptide having the amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO: 2,and a fragment of SEQ ID NO: 3 in conjunction with a suitablepharmaceutical carrier.

[0018] The invention further includes a purified antibody which binds toa polypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment ofSEQ ID NO: 1, a fragment of SEQ ID NO: 2, and a fragment of SEQ ID NO:3, as well as a purified agonist and a purified antagonist to thepolypeptide.

[0019] The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of the polypeptide havingan amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment ofSEQ ID NO: 2, and a fragment of SEQ ID NO: 3.

[0020] The invention also provides a method for treating or preventingan autoimmune disorder, the method comprising administering to a subjectin need of such treatment an effective amount of an antagonist of thepolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment ofSEQ ID NO: 1, a fragment of SEQ ID NO: 2, and a fragment of SEQ ID NO:3.

[0021] The invention also provides a method for treating or preventing aneuronal disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of thepolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment ofSEQ ID NO: 1, a fragment of SEQ ID NO: 2, and a fragment of SEQ ID NO:3.

[0022] The invention also provides a method for detecting apolynucleotide encoding the polypeptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, a fragment of SEQ ID NO: 2,and a fragment of SEQ ID NO: 3 in a biological sample containing nucleicacids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a fragment of SEQ ID NO: 1, afragment of SEQ ID NO: 2, and a fragment of SEQ ID NO: 3 to at least oneof the nucleic acids of the biological sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding the polypeptide in the biologicalsample. In one aspect, the nucleic acids of the biological sample areamplified by the polymerase chain reaction prior to the hybridizingstep.

DESCRIPTION OF THE INVENTION

[0023] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0024] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0025] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, vectors, and methodologies which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

[0026] DEFINITIONS

[0027] “UCEH,” as used herein, refers to the amino acid sequences ofsubstantially purified UCEH obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0028] The term “agonist,” as used herein, refers to a molecule which,when bound to UCEH, increases or prolongs the duration of the effect ofUCEH. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of UCEH.

[0029] An “allelic variant,” as this term is used herein, is analternative form of the gene encoding UCEH. Allelic variants may resultfrom at least one mutation in the nucleic acid sequence and may resultin altered mRNAs or in polypeptides whose structure or function may ormay not be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

[0030] “Altered” nucleic acid sequences encoding UCEH, as describedherein, include those sequences with deletions, insertions, orsubstitutions of different nucleotides, resulting in a polynucleotidethe same as UCEH or a polypeptide with at least one functionalcharacteristic of UCEH. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding UCEH,and improper or unexpected hybridization to allelic variants, with alocus other than the normal chromosomal locus for the polynucleotidesequence encoding UCEH. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent UCEH. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of UCEH is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

[0031] The terms “amino acid” or “amino acid sequence,” as used herein,refer to an oligopeptide, peptide, polypeptide, or protein sequence, ora fragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of UCEH which are preferablyabout 5 to about 15 amino acids in length, most preferably 14 aminoacids, and which retain some biological activity or immunologicalactivity of UCEH. Where “amino acid sequence” is recited herein to referto an amino acid sequence of a naturally occurring protein molecule,“amino acid sequence” and like terms are not meant to limit the aminoacid sequence to the complete native amino acid sequence associated withthe recited protein molecule.

[0032] “Amplification,” as used herein, relates to the production ofadditional copies of a nucleic acid sequence. Amplification is generallycarried out using polymerase chain reaction (PCR) technologies wellknown in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler(1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., pp. 1-5.)

[0033] The term “antagonist,” as it is used herein, refers to a moleculewhich, when bound to UCEH, decreases the amount or the duration of theeffect of the biological or immunological activity of UCEH. Antagonistsmay include proteins, nucleic acids, carbohydrates, antibodies, or anyother molecules which decrease the effect of UCEH.

[0034] As used herein, the term “antibody” refers to intact molecules aswell as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments,which are capable of binding the epitopic determinant. Antibodies thatbind UCEH polypeptides can be prepared using intact polypeptides orusing fragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0035] The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

[0036] The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

[0037] As used herein, the term “biologically active,” refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic UCEH, or ofany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0038] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

[0039] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding UCEH orfragments of UCEH may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g.,sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

[0040] “Consensus sequence,” as used herein, refers to a nucleic acidsequence which has been resequenced to resolve uncalled bases, extendedusing XL-PCR (Perkin Elmer, Norwalk, CT) in the 5′ and/or the 3′direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW Fragment Assemblysystem (GCG, Madison, WI). Some sequences have been both extended andassembled to produce the consensus sequence.

[0041] As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding UCEH, byNorthern analysis is indicative of the presence of nucleic acidsencoding UCEH in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding UCEH.

[0042] A “deletion,” as the term is used herein, refers to a change inthe amino acid or nucleotide sequence that results in the absence of oneor more amino acid residues or nucleotides.

[0043] The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0044] The term “similarity,” as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word “identity” may substitute for the word “similarity.” Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the second non-complementary target sequence.

[0045] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, Inc.,Madison Wisc.). The MEGALIGN program can create alignments between twoor more sequences according to different methods, e.g., the clustalmethod. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no similarity between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be counted orcalculated by other methods known in the art, e.g., the Jotun Heinmethod. (See, e.g., Hein, J. (1990) Methods Enzymol. 183: 626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

[0046] “Human artificial chromosomes” (HACs), as described herein, arelinear microchromosomes which may contain DNA sequences of about 6 kb to10 Mb in size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat. Genet. 15: 345-355.)

[0047] The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

[0048] “Hybridization,” as the term is used herein, refers to anyprocess by which a strand of nucleic acid binds with a complementarystrand through base pairing.

[0049] As used herein, the term “hybridization complex” refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀t or R₀t analysis) or formedbetween one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0050] The words “insertion” or “addition,” as used herein, refer tochanges in an amino acid or nucleotide sequence resulting in theaddition of one or more amino acid residues or nucleotides,respectively, to the sequence found in the naturally occurring molecule.

[0051] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0052] The term “microarray,” as used herein, refers to an arrangementof distinct polynucleotides arrayed on a substrate, e.g., paper, nylonor any other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

[0053] The terms “element” or “array element” as used herein in amicroarray context, refer to hybridizable polynucleotides arranged onthe surface of a substrate.

[0054] The term “modulate,” as it appears herein, refers to a change inthe activity of UCEH. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of UCEH.

[0055] The phrases “nucleic acid” or “nucleic acid sequence,” as usedherein, refer to a nucleotide, oligonucleotide, polynucleotide, or anyfragment thereof. These phrases also refer to DNA or RNA of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent the sense or the antisense strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material. In this context,“fragments” refers to those nucleic acid sequences which, whentranslated, would produce polypeptides retaining some functionalcharacteristic, e.g., antigenicity, or structural domain characteristic,e.g., ATP-binding site, of the full-length polypeptide.

[0056] The terms “operably associated” or “operably linked,” as usedherein, refer to functionally related nucleic acid sequences. A promoteris operably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

[0057] The term “oligonucleotide,” as used herein, refers to a nucleicacid sequence of at least about 6 nucleotides to 60 nucleotides,preferably about 15 to 30 nucleotides, and most preferably about 20 to25 nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

[0058] “Peptide nucleic acid” (PNA), as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least about 5 nucleotides in length linked to a peptide backboneof amino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen,P.E. et al. (1993) Anticancer Drug Des. 8: 53-63.)

[0059] The term “sample,” as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acids encoding UCEH,or fragments thereof, or UCEH itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

[0060] As used herein, the terms “specific binding” or “specificallybinding” refer to that interaction between a protein or peptide and anagonist, an antibody, or an antagonist. The interaction is dependentupon the presence of a particular structure of the protein, e.g., theantigenic determinant or epitope, recognized by the binding molecule.For example, if an antibody is specific for epitope “A,” the presence ofa polypeptide containing the epitope A, or the presence of freeunlabeled A, in a reaction containing free labeled A and the antibodywill reduce the amount of labeled A that binds to the antibody.

[0061] As used herein, the term “stringent conditions” refers toconditions which permit hybridization between polynucleotides and theclaimed polynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0062] For example, stringent salt concentration will ordinarily be lessthan about 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100μgmldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50 % formamide, and 200μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

[0063] The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0064] The term “substantially purified,” as used herein, refers tonucleic acid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

[0065] A “substitution,” as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0066] “Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

[0067] A “variant” of UCEH, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software.

[0068] THE INVENTION

[0069] The invention is based on the discovery of new humanubquitin-conjugating enzyme homologs (UCEH), the polynucleotidesencoding UCEH, and the use of these compositions for the diagnosis,treatment, or prevention of cancer, autoimmune disorders, and neuronaldisorders.

[0070] Nucleic acids encoding the UCEH-1 of the present invention werefirst identified in Incyte Clone 1728211 from the prostate tissue cDNAlibrary (PROSNOT14) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO: 4, was derivedfrom the following overlapping and/or extended nucleic acid sequences:Incyte Clones 1728211 (PROSNOT14), 2117351 (BRSTTUT02), 2849310(BRSTTIUT13), and 515286 (MMLRIDTO1).

[0071] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1. UCEH-1 is 250 aminoacids in length and has seven potential casein kinase phosphorylationsites at residues S₇, T₁₆, T₃₄, S₁₈₃, S₂₀₆, and S₂₃. In addition, UCEH-1has four potential protein kinase C phosphorylation sites at residuesS₆₅, T₉₁, T₉₅, and S₁₂₅, and a potential tyrosine kinase phosphorylationsite at residue Y_(b 41). PFAM analysis identifies UCEH-1 as anubiquitin-conjugating enzyme (UQ_con), with the region from residue 1through 149 receiving a score of 22 bits. BLOCKS analysis alsoidentifies UCEH-1 as an ubiquitin-conjugating enzyme (BL00183), with theregion from residue 24 through 76 receiving a score of 1137 and astrength of 1512. BLAST analysis indicates that UCEH-1has chemical andstructural similarity with Arabidopsis thaliana ubiquitin-conjugatingenzyme (GI 1707021). Northern analysis shows the expression of SEQ IDNO: 4 in various libraries, at least 36% of which are immortalized orcancerous and at least 50% of which involve immune response. Inaddition, 36% of the libraries showing expression of UCEH-1 were fromreproductive tissue, and 21% were from nervous tissue. Of particularnote is the expression of UCEH-1in spinal cord and pituitary libraries.A fragment of SEQ ID NO: 4 from about nucleotide 105 to about nucleotide165 is useful, e.g., as a hybridization probe.

[0072] Nucleic acids encoding the UCEH-2 of the present invention werefirst identified in Incyte Clone 1803905 from the ileum tissue cDNAlibrary (SINTNOT13) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO: 5, was derivedfrom the following overlapping and/or extended nucleic acid sequences:Incyte Clones 1803905 (SINTNOT13), 1521572 (BLADTUT04), 1869910(SKINBITO), 1901037 (BLADTUT06), and the shotgun sequence SAIA03806.

[0073] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2. UCEH-2 is 282 aminoacids in length and has an ubiquitin-conjugating enzyme active sitesignature sequence from W₈₂ through I₉₆. In addition, UCEH-2 has apotential N-glycosylation site at residue N₂₀₁, a potentialglycosaminoglycan attachment site at S₂₇₃, five potential casein kinaseII phosphorylation sites at residues S₁₉, T₃₁, T₅₁, T₁₇₂, and T₁₈₆,three potential protein kinase C phosphorylation sites at S9, T₇₄, andS₁₁₁, and two potential tyrosine kinase phosphorylation sites at Y₇₀ andY_(b 161). PFAM analysis identifies UCEH-2 as an ubiquitin-conjugatingenzyme (UQ_con) with the region from residue 5 through 167 receiving ascore of 264 bits. BLOCKS analysis also identifies UCEH-2 as anubiquitin-conjugating enzyme (BL00183), with the region from residue 41through 93 receiving a score of 1473 and a strength of 1512. BLASTanalysis indicates that UCEH-2 has chemical and structural similaritywith Oryctolagus cuniculus ubiquitin-conjugating enzyme E2 (GI 1381181).Northern analysis shows the expression of SEQ ID NO: 5 in variouslibraries, at least 47% of which are immortalized or cancerous and atleast 29% of which involve immune response. In addition, 31 % of thelibraries showing expression of UCEH-2 were from reproductive tissue,and 15% were from gastrointestinal tissue. Of particular note is theexpression of UCEH-2 in tumors of the colon and breast. A fragment ofSEQ ID NO: 5 from about nucleotide 480 to about nucleotide 540 isuseful, e.g., as a hybridization probe.

[0074] Nucleic acids encoding the UCEH-3 of the present invention werefirst identified in Incyte Clone 2792472 from the colon tumor tissuecDNA library (COLNTUT16) using a computer search, e.g., BLAST, for aminoacid sequence alignments. A consensus sequence, SEQ ID NO: 2, wasderived from the following overlapping and/or extended nucleic acidsequences: Incyte Clones 2792472 (COLNTUT16), 1575521 (LNODNOTO3),1444341 (THURNOT03), 915177 (BRSTNOT04), 1504874 (BRAITUT07), 981955(TONGTUT01), 2059855 (OVARNOT03), 568238 (MMLR3DT01), 1857985(PROSNOT18), and 1558539 and 1534471 (SPLNNOT04).

[0075] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 3. UCEH-3 is 318 aminoacids in length and has a potential amidation site at residue V₈₆. Inaddition, UCEH-3 has five potential N-glycosylation sites at residueN₁₉₀, N_(202, N) ₂₃₁, N₂₄₇, and N₂₈₀, a potential cAMP- andcGMP-dependent protein kinase phosphorylation site at residue S₂₆₆, ninepotential casein kinase II phosphorylation sites at residues S₅₁, T₁₂₁,T₁₃₃, S₁₆₅, S₁₇₀, T₁₉₅, S₁₉₇, S₂₀₆, and T₂₆₇, and six potential proteinkinase C phosphorylation sites at T₄₄, S₁₀₇, S₁₈₄, S₁₉₂, S₂₅₁, and S₂₆₁.PFAM analysis identifies UCEH-3 as an ubiquitin-conjugating enzyme(UQ_con) with the region from residue 6 through 151 receiving a score of71 bits. BLOCKS analysis also identifies UCEH-3 as anubiquitin-conjugating enzyme (BL00183), with the region from residue 41through 93 receiving a score of 1304 and a strength of 1512. BLASTanalysis indicates that UCEH-3 has chemical and structural similaritywith Caenorhabditis elegans ubiquitin-conjugating enzyme (GI 746510).Northern analysis shows the expression of SEQ ID NO: 6 in variouslibraries, at least 59% of which are immortalized or cancerous and atleast 46% of which involve immune response. In addition, 32% of thelibraries showing expression of UCEH-3 were from hematopoietic/immunetissue, 19% were from gastrointestinal tissue, and 19% were fromreproductive tissue. Of particular note is the expression of UCEH-3 intumors of the colon and brain. A fragment of SEQ ID NO: 6 from aboutnucleotide 32 to about nucleotide 92 is useful, e.g., as a hybridizationprobe.

[0076] The invention also encompasses UCEH variants. A preferred UCEHvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the UCEH amino acid sequence, and which contains at leastone functional or structural characteristic of UCEH.

[0077] The invention also encompasses polynucleotides which encode UCEH.In a particular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQID NO: 6, which encode UCEH.

[0078] A particular aspect of the invention encompasses a variant of apolynucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6, which has at least about 80%, morepreferably at least about 90%, and most preferably at least about 95%polynucleotide sequence identity to the selected polynucleotide. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of UCEH.

[0079] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding UCEH, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringUCEH, and all such variations are to be considered as being specificallydisclosed.

[0080] Although nucleotide sequences which encode UCEH and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring UCEH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding UCEH or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding UCEH and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0081] The invention also encompasses production of DNA sequences whichencode UCEH and UCEH derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingUCEH or any fragment thereof.

[0082] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, a fragment of SEQ ID NO: 4 a fragment of SEQ ID NO: 5, or afragment of SEQ ID NO: 6, under various conditions of stringency. (See,e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152: 399-407;Kimmel, A. R. (1987) Methods Enzymol. 152: 507- 51 1.)

[0083] Methods for DNA sequencing are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE DNA polymerase (US Biochemical Corp.,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System (GIBCO BRL, Gaithersburg, Md.). Preferably, theprocess is automated with machines such as the MICROLAB 2200 liquidtransfer system (Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200;MJ Research, Watertown, Mass.) and the ABI CATALYST and 373 and 377 DNAsequencers (Perkin Elmer).

[0084] The nucleic acid sequences encoding UCEH may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2: 318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16: 8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1: 111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19: 3055-306). Additionally, one may use PCR,nested primers, and PromoterFinder libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

[0085] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0086] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0087] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode UCEH may be cloned in recombinant DNAmolecules that direct expression of UCEH, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express UCEH.

[0088] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterUCEH-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0089] In another embodiment, sequences encoding UCEH may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp.Ser. 7: 215-223, and Horn, T. et al. (1980) Nucl. Acids Symp. Ser. 7:225-232.) Alternatively, UCEH itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269: 202-204.) Automated synthesis may beachieved using the ABI 431A peptide synthesizer (Perkin Elmer).Additionally, the amino acid sequence of UCEH, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0090] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182: 392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

[0091] In order to express a biologically active UCEH, the nucleotidesequences encoding UCEH or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding UCEH. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding UCEH. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding UCEH and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162.)

[0092] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding UCEHand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

[0093] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding UCEH. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0094] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding UCEH. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding UCEH can be achievedusing a multifunctional E. coli vector such as BLUESCRIPT (Stratagene)or PSPORT1 plasmid (GIBCO BRL). Ligation of sequences encoding UCEH intothe vector's multiple cloning site disrupts the lacZ gene, allowing acalorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264: 5503-5509. ) When large quantities of UCEH are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of UCEH may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

[0095] Yeast expression systems may be used for production of UCEH. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153: 516-54; Scorer, C. A. et al. (1994)Bio/Technology 12: 181-184.)

[0096] Plant systems may also be used for expression of UCEH.Transcription of sequences encoding UCEH may be driven viral promoters,e.g., the 35S and 19S promoters of CaMV used alone or in combinationwith the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.6: 307-311.) Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. etal. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by directDNA transformation or pathogen-mediated transfection. (See, e.g., Hobbs,S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

[0097] In mammalian cells, a number of viral-based expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding UCEH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses UCEH in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81: 3655-3659. ) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0098] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes.

[0099] For long term production of recombinant proteins in mammaliansystems, stable expression of UCEH in cell lines is preferred. Forexample, sequences encoding UCEH can be transformed into cell linesusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for about 1 to 2 days in enrichedmedia before being switched to selective media. The purpose of theselectable marker is to confer resistance to a selective agent, and itspresence allows growth and recovery of cells which successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be propagated using tissue culture techniques appropriate to thecell type.

[0100] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk or apr cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11: 223-232; andLowy, I. et al. (1980) Cell 22: 817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides neomycin and G-418; and als orpat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin, F. et al (1981)J. Mol. Biol. 150: 1-14; and Murry, supra.) Additional selectable geneshave been described, e.g., trpB and hisD, which alter cellularrequirements for metabolites. (See, e.g., Hartman, S. C. and R. C.Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-8051.) Visible markers,e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech, PaloAlto, Calif.), β glucuronidase and its substrate β-D-glucuronoside, orluciferase and its substrate luciferin may be used. These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55: 121-131.)

[0101] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding UCEH is inserted within a marker gene sequence, transformedcells containing sequences encoding UCEH can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding UCEH under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0102] In general, host cells that contain the nucleic acid sequenceencoding UCEH and that express UCEH may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0103] Immunological methods for detecting and measuring the expressionof UCEH using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on UCEH is preferred, but a competitive bindingassay may be employed. These and other assays are well known in the art.(See, e.g., Hampton, R. et al. (1990) Serological Methods, a LaboratoryManual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et al.(1997 and periodic supplements) Current Protocols in Immunology, GreenePub. Associates and Wiley-Interscience, New York, N.Y.; and Maddox, D.E. et al. (1983) J. Exp. Med. 158: 1211-1216).

[0104] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding UCEHinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding UCEH, or any fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byPharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wisc.), andU.S. Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules orlabels which may be used for ease of detection include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

[0105] Host cells transformed with nucleotide sequences encoding UCEHmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode UCEH may be designed to contain signal sequences which directsecretion of UCEH through a prokaryotic or eukaryotic cell membrane.

[0106] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Manassas, Va.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0107] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding UCEH may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric UCEHprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of UCEH activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the UCEH encodingsequence and the heterologous protein sequence, so that UCEH may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

[0108] In a further embodiment of the invention, synthesis ofradiolabeled UCEH may be achieved in vitro using the TNT rabbitreticulocyte lysate or wheat germ extract systems (Promega, Madison,Wisc.). These systems couple transcription and translation ofprotein-coding sequences operably associated with the T7, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, preferably ³⁵S-methionine.

[0109] Fragments of UCEH may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of UCEH may besynthesized separately and then combined to produce the full lengthmolecule.

[0110] THERAPEUTICS

[0111] Chemical and structural similarity exists among UCEH-1, UCEH-2,UCEH-3, and ubiquitin-conjugating enzymes from Arabidopsis thaliana (GI1707021), Oryctolagus cuniculus (GI 1381181), and Caenorhabditis elegans(GI 746510), respectively. PFAM and BLOCKS analysis identifies UCEH-1,UCEH-2, and UCEH-3 as ubiquitin-conjugating enzyme homologs. Inaddition, UCEH-1, UCEH-2, and UCEH-3 are expressed in cancerous andinflamed tissue, and are enriched in libraries from nervous tissue.Therefore, UCEH appears to play a role in cancer, autoimmune disorders,and neuronal disorders.

[0112] Therefore, in one embodiment, an antagonist of UCEH may beadministered to a subject to treat or prevent a cancer. Such a cancermay include, but is not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus. In oneaspect, an antibody which specifically binds UCEH may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express UCEH.

[0113] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding UCEH may be administered to a subject totreat or prevent a cancer including, but not limited to, those describedabove.

[0114] In another embodiment, an antagonist of UCEH may be administeredto a subject to treat or prevent an autoimmune disorder. Such a disordermay include, but is not limited to, acquired immunodeficiency syndrome(AIDS), Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma. Inone aspect, an antibody which specifically binds UCEH may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress UCEH.

[0115] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding UCEH may be administered to a subject totreat or prevent an autoimmune disorder including, but not limited to,those described above.

[0116] In yet another embodiment, an antagonist of UCEH may beadministered to a subject to treat or prevent a neuronal disorder. Sucha disorder may include, but is not limited to, akathesia, Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis, bipolar disorder,catatonia, cerebral neoplasms, dementia, depression, diabeticneuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, peripheral neuropathy, multiple sclerosis,neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpeticneuralgia, schizophrenia, and Tourette's disorder.. In one aspect, anantibody which specifically binds UCEH may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express UCEH.

[0117] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding UCEH may be administered to a subject totreat or prevent a neuronal disorder including, but not limited to,those described above.

[0118] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0119] An antagonist of UCEH may be produced using methods which aregenerally known in the art. In particular, purified UCEH may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind UCEH. Antibodies to UCEH may alsobe generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0120] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith UCEH or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0121] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to UCEH have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofUCEH amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0122] Monoclonal antibodies to UCEH may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256: 495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81: 31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; andCole, S. P. et al. (1984) Mol. Cell Biol. 62: 109-120.)

[0123] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81: 6851-6855; Neuberger, M. S. et al.(1984) Nature 312: 604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce UCEH-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)

[0124] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; and Winter, G. et al. (1991) Nature 349: 293-299.)

[0125] Antibody fragments which contain specific binding sites for UCEHmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246: 1275-1281.)

[0126] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between UCEH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering UCEH epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

[0127] In another embodiment of the invention, the polynucleotidesencoding UCEH, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding UCEH may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding UCEH. Thus, complementary molecules or fragments may be used tomodulate UCEH activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments can be designed from variouslocations along the coding or control regions of sequences encodingUCEH.

[0128] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding UCEH. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

[0129] Genes encoding UCEH can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding UCEH. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0130] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding UCEH. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. . et al. (1994) inHuber, B. E. nd B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementary sequenceor antisense molecule may also be designed to block translation of mRNAby preventing the transcript from binding to ribosomes.

[0131] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingUCEH.

[0132] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0133] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding UCEH. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0134] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0135] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15: 462-466.)

[0136] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0137] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of UCEH, antibodies to UCEH, and mimetics, agonists,antagonists, or inhibitors of UCEH. The compositions may be administeredalone or in combination with at least one other agent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0138] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0139] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0140] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0141] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0142] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0143] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0144] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0145] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0146] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0147] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0148] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of UCEH, such labeling wouldinclude amount, frequency, and method of administration.

[0149] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0150] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0151] A therapeutically effective dose refers to that amount of activeingredient, for example UCEH or fragments thereof, antibodies of UCEH,and agonists, antagonists or inhibitors of UCEH, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ ( (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0152] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0153] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0154] DIAGNOSTICS

[0155] In another embodiment, antibodies which specifically bind UCEHmay be used for the diagnosis of disorders characterized by expressionof UCEH, or in assays to monitor patients being treated with UCEH oragonists, antagonists, or inhibitors of UCEH. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for UCEH include methods whichutilize the antibody and a label to detect UCEH in human body fluids orin extracts of cells or tissues. The antibodies may be used with orwithout modification, and may be labeled by covalent or non-covalentattachment of a reporter molecule. A wide variety of reporter molecules,several of which are described above, are known in the art and may beused.

[0156] A variety of protocols for measuring UCEH, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of UCEH expression. Normal or standard valuesfor UCEH expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to UCEH under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of UCEH expressedin subject samples, control and disease, from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0157] In another embodiment of the invention, the polynucleotidesencoding UCEH may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof UCEH may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of UCEH, and tomonitor regulation of UCEH levels during therapeutic intervention.

[0158] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding UCEH or closely related molecules may be used to identifynucleic acid sequences which encode UCEH. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding UCEH, allelicvariants, or related sequences.

[0159] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of theUCEH encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of thesequences of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or fromgenomic sequences including promoters, enhancers, and introns of theUCEH gene.

[0160] Means for producing specific hybridization probes for DNAsencoding UCEH include the cloning of polynucleotide sequences encodingUCEH or UCEH derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

[0161] Polynucleotide sequences encoding UCEH may be used for thediagnosis of a disorder associated with expression of UCEH. Examples ofsuch a disorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus,autoimmune disorders such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma, andneuronal disorders such as akathesia, Alzheimer's disease, amnesia,amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebralneoplasms, dementia, depression, diabetic neuropathy, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease,peripheral neuropathy, multiple sclerosis, neurofibromatosis,Parkinson's disease, paranoid psychoses, postherpetic neuralgia,schizophrenia, and Tourette's disorder. The polynucleotide sequencesencoding UCEH may be used in Southern or Northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; in dipstick,pin, and ELISA assays; and in microarrays utilizing fluids or tissuesfrom patients to detect altered UCEH expression. Such qualitative orquantitative methods are well known in the art.

[0162] In a particular aspect, the nucleotide sequences encoding UCEHmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding UCEH may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding UCEH in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0163] In order to provide a basis for the diagnosis of a disorderassociated with expression of UCEH, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding UCEH, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0164] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0165] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0166] Additional diagnostic uses for oligonucleotides designed from thesequences encoding UCEH may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding UCEH, or a fragment of a polynucleotide complementary to thepolynucleotide encoding UCEH, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0167] Methods which may also be used to quantitate the expression ofUCEH include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and interpolating results from standardcurves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236.) Thespeed of quantitation of multiple samples may be accelerated by runningthe assay in an ELISA format where the oligomer of interest is presentedin various dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

[0168] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0169] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;Shalon, D. et al. (1995) PCT application W095/35505; Heller, R. A. etal. (1997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Heller, M. J. etal. (1997) U.S. Pat. No. 5,605,662.)

[0170] In another embodiment of the invention, nucleic acid sequencesencoding UCEH may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet. 7: 149-154.)

[0171] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) MolecularBiology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding UCEH on a physical chromosomalmap and a specific disorder, or a predisposition to a specific disorder,may help define the region of DNA associated with that disorder. Thenucleotide sequences of the invention may be used to detect differencesin gene sequences among normal, carrier, and affected individuals.

[0172] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336: 577-580.) The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0173] In another embodiment of the invention, UCEH, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between UCEHand the agent being tested may be measured.

[0174] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationW084/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with UCEH, orfragments thereof, and washed. Bound UCEH is then detected by methodswell known in the art. Purified UCEH can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0175] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding UCEHspecifically compete with a test compound for binding UCEH. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with UCEH.

[0176] In additional embodiments, the nucleotide sequences which encodeUCEH may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0177] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0178] I. Construction of cDNA Libraries

[0179] cDNA libraries were constructed from frozen tissue obtained fromthe small intestine (SINTNOT13), from a colon tumor (COLNTUT16), or froma prostate gland (PROSNOT14). The frozen tissue was homogenized andlysed in guanidinium isothiocyanate solution using a Polytron PT-3000homogenizer (Brinkmann Instruments, Westbury, N.Y.). The lysate wascentrifuged over a CsCl cushion to isolate RNA. Alternatively, RNA wasisolated using TRIzol reagent (Catalog #10296-028, GIBCO BRL,Gaithersburg, Md.), a monoplastic solution of phenol and guanidineisothiocyanate. The RNA was extracted with acid phenol, precipitatedwith sodium acetate and ethanol, resuspended in RNase-free water, andtreated with DNase. The RNA was re-extracted with acid phenol andre-precipitated with sodium acetate and ethanol. Poly(A+) RNA wasisolated using the OLIGOTEX kit (QIAGEN Inc, Chatsworth, Calif.). Thisprocedure may have been modified to accommodate the specific kits,plasmids, reagents, and machinery available at the time of eachlibrary's construction.

[0180] Poly(A+) RNA was used for cDNA synthesis and construction of thecDNA libraries according to the recommended protocols in the SUPERSCRIPTplasmid system (Catalog #18248-013, GIBCO BRL). The cDNAs werefractionated on a SEPHAROSE CL4B column (Catalog #275105-01, Pharmacia,Piscataway, N.J.), and those cDNAs exceeding 400 bp were ligated into anappropriate cDNA cloning vector, such as pINCY 1 (Incyte) or PSPORT 1(GIBCO BRL). The recombinant plasmids were subsequently transformed intoDH5a competent cells (Catalog #18258-012, GIBCO BRL).

[0181] II. Isolation and Sequencing of cDNA Clones

[0182] Plasmid DNA was released from the cells and purified using theR.E.A.L. Prep 96 Plasmid Kit (Catalog #26173, QIAGEN Inc). Therecommended protocol was employed except for the following changes: 1)the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog#22711, GIBCO BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%;2) after the cultures were incubated for 19 hours, the cells were lysedwith 0.3 ml of lysis buffer; and 3) following isopropanol precipitation,the plasmid DNA pellets were each resuspended in 0.1 ml of distilledwater. The DNA samples were stored at 4° C.

[0183] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94: 441f), using a MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nv.) in combination with Peltier thermal cyclers(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems 377DNA sequencing systems.

[0184] III. Similarity Searching of cDNA Clones and Their DeducedProteins

[0185] The nucleotide sequences and/or amino acid sequences of theSequence Listing were used to query sequences in the GenBank, SwissProt,BLOCKS, and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofsimilarity using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36: 290-300; and Altschul et al.(1990) J. Mol. Biol. 215: 403-410.)

[0186] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. (See, e.g., Smith, T. etal. (1992) Protein Engineering 5: 35-51.) The sequences disclosed inthis application have lengths of at least 49 nucleotides and have nomore than 12% uncalled bases (where N is recorded rather than A, C, G,or T).

[0187] The BLAST approach searched for matches between a query sequenceand a database sequence. BLAST evaluated the statistical significance ofany matches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸for peptides.

[0188] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam), and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for similarity.

[0189] Additionally, sequences identified from cDNA libraries may beanalyzed to identify those gene sequences encoding conserved proteinmotifs using an appropriate analysis program, e.g., BLOCKS. BLOCKS is aweighted matrix analysis algorithm based on short amino acid segments,or blocks, compiled from the PROSITE database. (Bairoch, A. et al.(1997) Nucleic Acids Res. 25: 217-221.) The BLOCKS algorithm is usefulfor classifying genes with unknown functions. (Henikoff S. And HenikoffG. J., Nucleic Acids Research (1991) 19: 6565-6572.) Blocks, which are3-60 amino acids in length, correspond to the most highly conservedregions of proteins. The BLOCKS algorithm compares a query sequence witha weighted scoring matrix of blocks in the BLOCKS database. Blocks inthe BLOCKS database are calibrated against protein sequences with knownfunctions from the SWISS-PROT database to determine the stochasticdistribution of matches. Similar databases such as PRINTS, a proteinfingerprint database, are also searchable using the BLOCKS algorithm.(Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37: 417-424.)PRINTS is based on non-redundant sequences obtained from sources such asSWISS-PROT, GenBank, PIR, and NRL-3D.

[0190] The BLOCKS algorithm searches for matches between a querysequence and the BLOCKS or PRINTS database and evaluates the statisticalsignificance of any matches found. Matches from a BLOCKS or PRINTSsearch can be evaluated on two levels, local similarity and globalsimilarity. The degree of local similarity is measured by scores, andthe extent of global similarity is measured by score ranking andprobability values. A score of 1000 or greater for a BLOCKS match ofhighest ranking indicates that the match falls within the 0.5 percentilelevel of false positives when the matched block is calibrated againstSWISS-PROT. Likewise, a probability value of less than 1.0×10⁻³indicates that the match would occur by chance no more than one time inevery 1000 searches. Only those matches with a cutoff score of 1000 orgreater and a cutoff probability value of 1.0×10³¹ ³ or less areconsidered in the functional analyses of the protein sequences in theSequence Listing.

[0191] Nucleic and amino acid sequences of the Sequence Listing may alsobe analyzed using PFAM. PFAM is a Hidden Markov Model (HMM) basedprotocol useful in protein family searching. HMM is a probalisticapproach which analyzes consensus primary structures of gene families.(See, e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6: 361-365.)

[0192] The PFAM database contains protein sequences of 527 proteinfamilies gathered from publicly available sources, e.g., SWISS-PROT andPROSITE. PFAM searches for well characterized protein domain familiesusing two high-quality alignment routines, seed alignment and fullalignment. (See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins 28:405-420.) The seed alignment utilizes the hmmls program, a program thatsearches for local matches, and a non-redundant set of the PFAMdatabase. The full alignment utilizes the hmmfs program, a program thatsearches for multiple fragments in long sequences, e.g., repeats andmotifs, and all sequences in the PFAM database. A result or score of 100“bits” can signify that it is 2¹⁰⁰-fold more likely that the sequence isa true match to the model or comparison sequence. Cutoff scores whichrange from 10 to 50 bits are generally used for individual proteinfamilies using the SWISS-PROT sequences as model or comparisonsequences.

[0193] Two other algorithms, SIGPEPT and TM, both based on the HMMalgorithm described above (see, e.g., Eddy, supra; and Sonnhammer,supra), identify potential signal sequences and transmembrane domains,respectively. SIGPEPT was created using protein sequences having signalsequence annotations derived from SWISS-PROT. It contains about 1413non-redundant signal sequences ranging in length from 14 to 36 aminoacid residues. TM was created similarly using transmembrane domainannotations. It contains about 453 non-redundant transmembrane sequencesencompassing 1579 transmembrane domain segments. Suitable HMM modelswere constructed using the above sequences and were refined with knownSWISS-PROT signal peptide sequences or transmembrane domain sequencesuntil a high correlation coefficient, a measurement of the correctnessof the analysis, was obtained. Using the protein sequences from theSWISS-PROT database as a test set, a cutoff score of 11 bits, asdetermined above, correlated with 91-94% true-positives and about 4.1%false-positives, yielding a correlation coefficient of about 0.87-0.90for SIGPEPT. A score of 11 bits for TM will typically give the followingresults: 75% true positives; 1.72% false positives; and a correlationcoefficient of 0.76. Each search evaluates the statistical significanceof any matches found and reports only those matches that score at least11 bits.

[0194] IV. Northern Analysis

[0195] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; and 3Ausubel, supra, ch. 4 and 16.)

[0196] Analogous computer techniques applying BLAST are used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Pharmaceuticals). This analysis ismuch faster than multiple membrane-based hybridizations. In addition,the sensitivity of the computer search can be modified to determinewhether any particular match is categorized as exact or similar.

[0197] The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0198] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

[0199] The results of Northern analysis are reported as a list oflibraries in which the transcript encoding UCEH occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0200] V. Extension of UCEH Encoding Polynucleotides

[0201] The nucleic acid sequences of Incyte Clones 1728211, 1803905, and2792472 were used to design oligonucleotide primers for extendingpartial nucleotide sequences to full length. For each nucleic acidsequence, one primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06 primeranalysis software (National Biosciences, Plymouth, Minn.), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

[0202] Selected human cDNA libraries (GIBCO BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0203] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. PCR was performed using the Peltier thermalcycler (PTC200; M. J. Research, Watertown, Mass.), beginning with 40pmol of each primer and the recommended concentrations of all othercomponents of the kit, with the following parameters: Step 1 94° C. for1 min (initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6min Step 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7min Step 7 Repeat steps 4 through 6 for an additional 15 cycles Step 894° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 minStep 11 Repeat steps 8 through 10 for an additional 12 cycles Step 1272° C. for 8 min Step 13  4° C. (and holding)

[0204] A 5μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

[0205] After ethanol precipitation, the products were redissolved in13μl of ligation buffer, T4-DNA ligase (15 units) and 1μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin(2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2×carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1: 10 with water, 5 μl from each sample was transferred into aPCR array.

[0206] For PCR amplification, 18μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4for an additional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0207] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0208] In like manner, the nucleotide sequences of SEQ ID NO: 4, SEQ IDNO: 5, and SEQ ID NO: 6 are used to obtain 5′ regulatory sequences usingthe procedure above, oligonucleotides designed for 5 extension, and anappropriate genomic library.

[0209] VI. Labeling and Use of Individual Hybridization Probes

[0210] Hybridization probes derived from of SEQ ID NO: 4, SEQ ID NO: 5,and SEQ ID NO: 6 are employed to screen cDNAs, genomic DNAs, or mRNAs.Although the labeling of oligonucleotides, consisting of about 20 basepairs, is specifically described, essentially the same procedure is usedwith larger nucleotide fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 software (NationalBiosciences) and labeled by combining 50 pmol of each oligomer, 250 μCiof [γ−³²P] adenosine triphosphate (Amersham, Chicago, Ill.), and T4polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeledoligonucleotides are substantially purified using a SEPHADEX G-25superfine size exclusion dextran bead column (Pharmacia & Upjohn,Kalamazoo, Mich.). An aliquot containing 10⁷ counts per minute of thelabeled probe is used in a typical membrane-based hybridization analysisof human genomic DNA digested with one of the following endonucleases:Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN, Boston,Mass.).

[0211] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, NH). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 × salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, hybridization patternsare compared visually.

[0212] VII. Microarrays

[0213] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0214] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments suitablefor hybridization can be selected using software well known in the artsuch as LASERGENE. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270: 467-470; andShalon, D. et al. (1996) Genome Res. 6: 639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

[0215] VIII. Complementary Polynucleotides

[0216] Sequences complementary to the UCEH-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring UCEH. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 primeranalysis software and the coding sequence of UCEH. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the UCEH-encoding transcript.

[0217] IX. Expression of UCEH

[0218] Expression and purification of UCEH is achieved using bacterialor virus-based expression systems. For expression of UCEH in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express UCEH uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof UCEH in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding UCEH by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227; Sandig, V. et al.(1996) Hum. Gene Ther. 7: 1937-1945.)

[0219] In most expression systems, UCEH is synthesized as a fusionprotein with, e.g., glutathione S-transferase (GST) or a peptide epitopetag, such as FLAG or 6-His, permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Pharmacia, Piscataway, N.J). Following purification, the GST moiety canbe proteolytically cleaved from UCEH at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins (QIAGENInc, Chatsworth, Calif.). Methods for protein expression andpurification are discussed in Ausubel, F. M. et al. (1995 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., ch 10, 16. Purified UCEH obtained by these methods canbe used directly in the following activity assay.

[0220] X. Demonstration of UCEH Activity

[0221] UCEH activity is demonstrated by the formation of di-ubiquitinconjugates from free ubiquitin (van Nocker et al. supra). UCEH isincubated together with 75 pmol ¹²⁵I-labeled ubiquitin, 20 nM wheat E1,2 mM Mg ATP, 0.1 mM dithiothreitol, and 50 mM Tris-HCI, pH 8.0. Thereaction is incubated for 2 minutes at 4° C, and the di-ubiquitinproduct separated from free ubiquitin by polyacrylamide gelelectrophoresis. Di-ubiquitin is visualized by autoradiography, removedfrom the gel, and counted in a gamma radioisotope counter. The amount ofdi-ubiquitin formed in the reaction is proportional to the activity ofUCEH in the assay.

[0222] XI. Functional Assays

[0223] UCEH function is assessed by expressing the sequences encodingUCEH at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include PCMV SPORT plasmid (Life Technologies, Gaithersburg, Md.)and PCR 3.1 plasmid (Invitrogen, Carlsbad, Calif., both of which containthe cytomegalovirus promoter. 5-10 μg of recombinant vector aretransiently transfected into a human cell line, preferably ofendothelial or hematopoietic origin, using either liposome formulationsor electroporation. 1-2 μg of an additional plasmid containing sequencesencoding a marker protein are co-transfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP) (Clontech, Palo Alto, Calif.), CD64, or aCD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York, N.Y.

[0224] The influence of UCEH on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodingUCEH and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding UCEH and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

[0225] XII. Production of UCEH Specific Antibodies

[0226] UCEH substantially purified using polyacrylamide gelelectrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182: 488-495), or other purification techniques, is used toimmunize rabbits and to produce antibodies using standard protocols.

[0227] Alternatively, the UCEH amino acid sequence is analyzed usingLASERGENE software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel supra, ch. 11.)

[0228] Typically, oligopeptides 15 residues in length are synthesizedusing an Applied Biosystems peptide synthesizer Model 431A usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

[0229] XIII. Purification of Naturally Occurring UCEH Using SpecificAntibodies

[0230] Naturally occurring or recombinant UCEH is substantially purifiedby immunoaffinity chromatography using antibodies specific for UCEH. Animmunoaffinity column is constructed by covalently coupling anti-UCEHantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0231] Media containing UCEH are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of UCEH (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/UCEH binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and UCEHis collected. XIV. Identification of Molecules Which Interact with UCEH

[0232] UCEH, or biologically active fragments thereof, are labeled with1251 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133: 529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled UCEH, washed, and anywells with labeled UCEH complex are assayed. Data obtained usingdifferent concentrations of UCEH are used to calculate values for thenumber, affinity, and association of UCEH with the candidate molecules.

[0233] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 6 250 amino acids amino acid single linear PROSNOT14 1728211 1 Met AlaLeu Leu Ala Thr Ser Leu Pro Glu Gly Ile Met Val Lys 5 10 15 Thr Phe GluAsp Arg Met Asp Leu Phe Ser Ala Leu Ile Lys Gly 20 25 30 Pro Thr Arg ThrPro Tyr Glu Asp Gly Leu Tyr Leu Phe Asp Ile 35 40 45 Gln Leu Pro Asn IleTyr Pro Ala Val Pro Pro His Phe Cys Tyr 50 55 60 Leu Ser Gln Cys Ser GlyArg Leu Asn Pro Asn Leu Tyr Asp Asn 65 70 75 Gly Lys Val Cys Val Ser LeuLeu Gly Thr Trp Ile Gly Lys Gly 80 85 90 Thr Glu Arg Trp Thr Ser Lys SerSer Leu Leu Gln Val Leu Ile 95 100 105 Ser Ile Gln Gly Leu Ile Leu ValAsn Glu Pro Tyr Tyr Asn Glu 110 115 120 Ala Gly Phe Asp Ser Asp Arg GlyLeu Gln Glu Gly Tyr Glu Asn 125 130 135 Ser Arg Cys Tyr Asn Glu Met AlaLeu Ile Arg Val Val Gln Ser 140 145 150 Met Thr Gln Leu Val Arg Arg ProPro Glu Val Phe Glu Gln Glu 155 160 165 Ile Arg Gln His Phe Ser Thr GlyGly Trp Arg Leu Val Asn Arg 170 175 180 Ile Glu Ser Trp Leu Glu Thr HisAla Leu Leu Glu Lys Ala Gln 185 190 195 Ala Leu Pro Asn Gly Val Pro LysAla Ser Ser Ser Pro Glu Pro 200 205 210 Pro Ala Val Ala Glu Leu Ser AspSer Gly Gln Gln Glu Pro Glu 215 220 225 Asp Gly Gly Pro Ala Pro Gly GluAla Ser Gln Gly Ser Asp Ser 230 235 240 Glu Gly Gly Ala Gln Gly Leu AlaPhe Ser 245 250 282 amino acids amino acid single linear SINTNOT131803905 2 Met Ala Gln Gln Gln Met Thr Ser Ser Gln Lys Ala Leu Met Leu 510 15 Glu Leu Lys Ser Leu Gln Glu Glu Pro Val Glu Gly Phe Arg Ile 20 2530 Thr Leu Val Asp Glu Ser Asp Leu Tyr Asn Trp Glu Val Ala Ile 35 40 45Phe Gly Leu Pro Asn Thr Leu Tyr Glu Gly Gly Tyr Phe Lys Ala 50 55 60 HisIle Lys Phe Pro Ile Asp Tyr Pro Tyr Ser Pro Pro Thr Phe 65 70 75 Arg PheLeu Thr Lys Met Trp His Pro Asn Ile Tyr Glu Asn Gly 80 85 90 Asp Val CysIle Ser Ile Leu His Pro Pro Val Asp Asp Pro Gln 95 100 105 Ser Gly GluLeu Pro Ser Glu Arg Trp Asn Pro Thr Gln Asn Val 110 115 120 Arg Thr IleLeu Leu Ser Val Ile Ser Leu Leu Asn Glu Pro Asn 125 130 135 Thr Phe SerPro Ala Asn Val Asp Ala Ser Val Met Phe Arg Lys 140 145 150 Trp Arg AspSer Lys Gly Lys Asp Lys Glu Tyr Ala Glu Ile Ile 155 160 165 Arg Lys GlnVal Ser Ala Thr Lys Ala Glu Ala Glu Lys Asp Gly 170 175 180 Val Lys ValPro Thr Thr Leu Ala Glu Tyr Cys Ile Lys Thr Lys 185 190 195 Val Pro SerAsn Asp Asn Ser Ser Asp Leu Leu Tyr Asp Asp Leu 200 205 210 Tyr Asp AspAsp Ile Asp Asp Glu Asp Glu Glu Glu Glu Asp Ala 215 220 225 Asp Cys TyrAsp Asp Asp Asp Ser Gly Met Arg Ser Arg Asp Val 230 235 240 Leu Leu GlnCys Pro Cys Thr Ala Leu Pro Ser Gln Ala Lys Gly 245 250 255 Arg Gly AlaSer Gly Asp Leu Ala Met Ala Pro Gln Gln Lys Pro 260 265 270 Ile His SerGly Trp Gly Asn Thr His Ser Ser Cys 275 280 318 amino acids amino acidsingle linear COLNTUT16 2792472 3 Met Glu Thr Arg Tyr Asn Leu Lys SerPro Ala Val Lys Arg Leu 5 10 15 Met Lys Glu Ala Ala Glu Leu Lys Asp ProThr Asp His Tyr His 20 25 30 Ala Gln Pro Leu Glu Asp Asn Leu Phe Glu TrpHis Phe Thr Val 35 40 45 Arg Gly Pro Pro Asp Ser Asp Phe Asp Gly Gly ValTyr His Gly 50 55 60 Arg Ile Val Leu Pro Pro Glu Tyr Pro Met Lys Pro ProSer Ile 65 70 75 Ile Leu Leu Thr Ala Asn Gly Arg Phe Glu Val Gly Lys LysIle 80 85 90 Cys Leu Ser Ile Ser Gly His His Pro Glu Thr Trp Gln Pro Ser95 100 105 Trp Ser Ile Arg Thr Ala Leu Leu Ala Ile Ile Gly Phe Met Pro110 115 120 Thr Lys Gly Glu Gly Ala Ile Gly Ser Leu Asp Tyr Thr Pro Glu125 130 135 Glu Arg Arg Ala Leu Ala Lys Lys Ser Gln Asp Phe Cys Cys Glu140 145 150 Gly Cys Gly Ser Ala Met Lys Asp Val Leu Leu Pro Leu Lys Ser155 160 165 Gly Ser Asp Ser Ser Gln Ala Asp Gln Glu Ala Lys Glu Leu Ala170 175 180 Arg Gln Ile Ser Phe Lys Ala Glu Val Asn Ser Ser Gly Lys Thr185 190 195 Ile Ser Glu Ser Asp Leu Asn His Ser Phe Ser Leu Thr Asp Leu200 205 210 Gln Asp Asp Ile Pro Thr Thr Phe Gln Gly Ala Thr Ala Ser Thr215 220 225 Ser Tyr Gly Leu Gln Asn Ser Ser Ala Ala Ser Phe His Gln Pro230 235 240 Thr Gln Pro Val Ala Lys Asn Thr Ser Met Ser Pro Arg Gln Arg245 250 255 Arg Ala Gln Gln Gln Ser Gln Arg Arg Leu Ser Thr Ser Pro Asp260 265 270 Val Ile Gln Gly His Gln Pro Arg Asp Asn His Thr Asp His Gly275 280 285 Gly Ser Ala Val Leu Ile Val Ile Leu Thr Leu Ala Leu Ala Ala290 295 300 Leu Ile Phe Arg Arg Ile Tyr Leu Ala Asn Glu Tyr Ile Phe Asp305 310 315 Phe Glu Leu 1006 base pairs nucleic acid single linearPROSNOT14 1728211 4 AGGTCTTCTC CGTACTGGAG TTTGCACCCT CAAATCATTCTTTTAAGAAA ATTGAGTTCC 60 AGCCTCCAGA AGCCAAGAAG TTCTTCAGCA CAGTGCGGAAGGAGATGGCG CTGCTGGCTA 120 CCTCACTGCC TGAGGGCATC ATGGTCAAGA CTTTTGAAGATAGAATGGAC CTCTTCTCAG 180 CTCTCATCAA GGGCCCCACT CGAACCCCCT ACGAGGATGGCCTCTACTTG TTTGACATCC 240 AGCTCCCCAA CATCTACCCA GCCGTGCCCC CCCACTTCTGCTACCTCTCC CAATGCAGTG 300 GCCGCCTGAA CCCCAACCTG TATGACAATG GGAAGGTGTGTGTCAGCCTC CTGGGCACCT 360 GGATTGGAAA GGGGACAGAG AGGTGGACAA GCAAGTCCAGCCTTCTCCAG GTGCTCATCT 420 CCATCCAAGG TCTGATCCTG GTAAATGAAC CATACTACAACGAAGCCGGC TTCGACAGTG 480 ACCGAGGCCT GCAGGAAGGC TATGAAAACA GTCGCTGTTACAATGAGATG GCGCTGATCC 540 GCGTGGTGCA GTCCATGACC CAGCTGGTGC GGCGGCCCCCCGAGGTCTTT GAGCAGGAGA 600 TCAGGCAACA CTTTAGCACT GGTGGCTGGC GGCTGGTGAACCGTATCGAG TCCTGGCTGG 660 AAACCCATGC CCTGCTGGAG AAGGCCCAGG CACTGCCCAACGGGGTGCCC AAGGCCAGCA 720 GCTCGCCAGA GCCCCCAGCT GTAGCCGAGC TGTCAGACTCCGGCCAACAA GAACCTGAGG 780 ATGGAGGGCC AGCCCCAGGA GAGGCCTCCC AGGGCTCAGACTCAGAGGGC GGTGCCCAGG 840 GCCTGGCCTT CAGCTAGCAG GGACCACACA GACCAGACTTCGGAGACCGC ACCAGACGCA 900 TCGGTGCCAC CCAGTGTGAA ACCCAAAGAA GCGGAGAAAGAGCNTTAAGA GCTACCGGAG 960 CTTCTTACCT GAGAAGAGTG GCTACCCTGA CATCGGCTTCCCCCTC 1006 2067 base pairs nucleic acid single linear SINTNOT13 18039055 TTTTNTGTGT GTGCNCCCNG TNNGTGNCCC TNCTANGTGG TGGGNTTTGT TGTNGGTGTC 60TTCCTCTGTT TNTNTTTNCC TTGCCATTGC CTCAGNCTTC CNGTCCTCNT NNNCCNNTTT 120TGNGCNCCCC CCNATGTCCN TGCGCCCTAG NNGTTTGGNN CNCCCCCAAA GNTCTCNGNC 180CTNNCTGAAA GGGNNCCCCC CTNNGAAAAG GCCTNCTCGG NAATTGGNGT AAAAANATCC 240CCCNTNGCCA AGGGNGGGNG GGGAAAACCC CGGGTTTNGG AAAGGGGGCN TTTTNCCGNG 300GNNATTNCCC CCCCTGTTGG GTNTGGNACC GGNNTTCCCC NGANCCTNTT TTAACNAANC 360TTGGGGGAAG GTTTNNCCAA NTTNTTTCGG GACTTCCCCC CAACACNCNT TTTANNGGAA 420NGGCGGNCNT ATTTCTTCGG CCGAGGGTTA CATTCTGATG TTGGAGCGCC GCCGCCGCGA 480TGGCCCAGCA GCAGATGACC AGCTCGCAGA AGGCCCTGAT GCTCGAGCTG AAATCCCTGC 540AGGAGGAACC GGTGGAGGGC TTCCGGATCA CCCTGGTGGA CGAGTCCGAC CTCTACAACT 600GGGAGGTGGC CATCTTCGGA CTCCCCAACA CCCTCTACGA AGGCGGCTAC TTCAAGGCGC 660ATATTAAATT TCCTATTGAC TACCCCTATT CACCACCTAC CTTCAGATTC TTGACCAAAA 720TGTGGCACCC CAACATTTAT GAGAATGGAG ATGTATGCAT TTCGATTCTT CATCCGCCTG 780TAGATGACCC ACAGAGTGGA GAACTGCCTT CTGAAAGGTG GAATCCTACT CAGAATGTGA 840GGACTATCCT ATTAAGTGTA ATCTCACTGC TTAATGAGCC CAACACCTTC TCCCCAGCCA 900ATGTCGATGC TTCAGTTATG TTCAGGAAAT GGAGAGACAG TAAAGGAAAA GACAAAGAAT 960ATGCTGAAAT TATTAGGAAA CAAGTTTCAG CCACTAAGGC CGAAGCAGAA AAGGATGGAG 1020TGAAGGTCCC CACAACCCTG GCGGAATACT GCATCAAAAC TAAAGTGCCT TCCAATGACA 1080ACAGCTCAGA TTTGCTTTAC GACGACTTGT ATGATGACGA CATTGATGAT GAAGATGAGG 1140AGGAGGAAGA TGCCGACTGT TATGATGATG ATGATTCTGG GATGAGGAGT CGTGACGTGC 1200TCCTTCAGTG CCCCTGTACT GCCCTGCCAT CTCAGGCCAA AGGGAGGGGA GCAAGTGGGG 1260ACCTGGCCAT GGCCCCTCAG CAAAAACCTA TTCACAGCGG GTGGGGAAAC ACACACAGCT 1320CCTGCTGACT CCCCTTATGG ATCTCAGTTT GCTCCTTTTT ATGGACCTTT AATGGAGAGA 1380GAGTAACCCT CCACAGAATG TCTGAATTCT TGCATTCTTT ACCCTTCCAT CACTATATTG 1440ATTCTTTTTT TAAAAAATAT GAACCCAAAC TCCCGCCTCA CTTCGTCTCT ACAGAATGTT 1500CACAGCAAAA CACGTTTGGT CTGTTTTTAG ATTCTTGAAG AATTCAATAG TCTTTCAAGA 1560TGTTTAATGT GTTTAAAGCT GGGAACCTGT TGGGAGTTCA CAAGTGCTGC ATATACTGGG 1620TAGCAAAAGA AAATGGAAAA AAACCCACAA AACAAACTTT AAAAAAAAAA AAAAACAAAT 1680TTGCCAAGGT TTAGCTGCTC ATTTACATTA GTGTGTGTGC ATTCGTTCAG CCCCATGGTG 1740GTGAATTCTG TTTCTTTCCT TTCCTAAGGC TGGGACATGG TGGGCATCAG GGACTTTGTG 1800CTAAGCCTGA TGAAATGTGC TCCTTCAATC TCCATGAAAC CATCGTAACA TGGAGGCCTC 1860AGCTGCTCTG AGGAGAGAAA TCAGACTTTG TTTTTTGAAA TCGATTGGGA TCGAAAGCCT 1920GAAATAAATA TTCATACTTT CCATAGTCCA CCCAAAATGA GAAAGGAGGA GAAAAAAAAA 1980AAGGGGGGGG CGCCGGCCTA GTGACCCCTG TCGACCCGGG AATTAAATTC CGGACCGGGA 2040CCTGCAGGGG TGTACCAGGT TTTCCCT 2067 1067 base pairs nucleic acid singlelinear COLNTUT16 2792472 6 CGGGAGGCCG GAGCCAAGCC AGCGACCCAC CATGGAGACCCGCTACAACC TGAAGAGTCC 60 GGCTGTTAAA CGTTTAATGA AAGAAGCGGC AGAATTGAAAGATCCAACAG ATCATTACCA 120 TGCGCAGCCT TTAGAGGATA ACCTTTTTGA ATGGCACTTCACGGTTAGAG GGCCCCCAGA 180 CTCCGATTTT GATGGAGGAG TTTATCACGG GCGGATAGTACTGCCACCAG AGTATCCCAT 240 GAAACCACCA AGCATTATTC TCCTAACGGC TAATGGTCGATTTGAAGTGG GCAAGAAAAT 300 CTGTTTGAGC ATCTCAGGCC ATCATCCTGA AACTTGGCAGCCTTCGTGGA GTATAAGGAC 360 AGCATTATTA GCCATCATTG GGTTTATGCC AACAAAAGGAGAGGGAGCCA TAGGTTCTCT 420 AGATTACACT CCTGAGGAAA GAAGAGCACT TGCCAAAAAATCACAAGATT TCTGTTGTGA 480 AGGATGTGGC TCTGCCATGA AGGATGTCCT GTTGCCTTTAAAATCTGGAA GCGATTCAAG 540 CCAAGCTGAC CAAGAAGCCA AAGAACTGGC TAGGCAAATAAGCTTTAAGG CAGAAGTCAA 600 TTCATCTGGA AAGACTATCT CTGAGTCAGA CTTAAACCACTCTTTTTCAC TAACTGATTT 660 ACAAGATGAT ATACCTACAA CATTCCAGGG TGCTACGGCCAGTACATCGT ACGGACTCCA 720 GAATTCCTCA GCAGCATCCT TTCATCAACC TACCCAACCTGTAGCTAAGA ATACCTCCAT 780 GAGCCCTCGA CAGCGCCGGG CCCAGCAGCA GAGTCAGAGAAGGTTGTCTA CTTCACCAGA 840 TGTAATCCAG GGCCACCAGC CAAGAGACAA CCACACTGATCATGGTGGGT CAGCTGTACT 900 GATTGTCATC CTGACTTTGG CATTGGCAGC TCTTATATTCCGACGAATAT ATCTGGCAAA 960 CGAATACATA TTTGACTTTG AGTTATAATA TGGTTTTGTGACTTATGAGC TGTGACTCAA 1020 CTGCTTCATT AAACATTCTG CATTGGGTAT AATCTAAAAAAAAAAAA 1067

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-3, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO: 1-3, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-3.
 2. An isolated polypeptide of claim 1 selected from the groupconsisting of SEQ ID NO: 1-3.
 3. An isolated polynucleotide encoding apolypeptide of claim
 1. 4. An isolated polynucleotide encoding apolypeptide of claim
 2. 5. An isolated polynucleotide of claim 4selected from the group consisting of SEQ ID NO: 4-6.
 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. A transgenic organism comprising arecombinant polynucleotide of claim
 6. 9. A method of producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 11. An isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO: 4-6, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ ID NO:4-6, c) a polynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 12. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 11. 13. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 11, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 14. A method of claim 13, wherein the probe comprises atleast 60 contiguous nucleotides.
 15. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 11, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 16. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 17. Acomposition of claim 16, wherein the polypeptide has an amino acidsequence selected from the group consisting of SEQ ID NO: 1-3.
 18. Amethod for treating a disease or condition associated with decreasedexpression of functional UCEH, comprising administering to a patient inneed of such treatment the composition of claim
 16. 19. A method ofscreening a compound for effectiveness as an agonist of a polypeptide ofclaim 1, the method comprising: a) exposing a sample comprising apolypeptide of claim 1 to a compound, and b) detecting agonist activityin the sample.
 20. A composition comprising an agonist compoundidentified by a method of claim 19 and a pharmaceutically acceptableexcipient.
 21. A method for treating a disease or condition associatedwith decreased expression of functional UCEH, comprising administeringto a patient in need of such treatment a composition of claim
 20. 22. Amethod of screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample.
 23. A composition comprising anantagonist compound identified by a method of claim 22 and apharmaceutically acceptable excipient.
 24. A method for treating adisease or condition associated with overexpression of functional UCEH,comprising administering to a patient in need of such treatment acomposition of claim
 23. 25. A method of screening for a compound thatspecifically binds to the polypeptide of claim 1, the method comprising:a) combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 26. A method ofscreening for a compound that modulates the activity of the polypeptideof claim 1, the method comprising: a) combining the polypeptide of claim1 with at least one test compound under conditions permissive for theactivity of the polypeptide of claim 1, b) assessing the activity of thepolypeptide of claim 1 in the presence of the test compound, and c)comparing the activity of the polypeptide of claim 1 in the presence ofthe test compound with the activity of the polypeptide of claim 1 in theabsence of the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 27. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 28. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 11 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 11 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 29. Adiagnostic test for a condition or disease associated with theexpression of UCEH in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 10, underconditions suitable for the antibody to bind the polypeptide and form anantibody: polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 30. The antibody of claim 10, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 31. Acomposition comprising an antibody of claim 10 and an acceptableexcipient.
 32. A method of diagnosing a condition or disease associatedwith the expression of UCEH in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 31. 33. Acomposition of claim 31, wherein the antibody is labeled.
 34. A methodof diagnosing a condition or disease associated with the expression ofUCEH in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 33. 35. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 10,the method comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence selected from the group consisting of SEQ ID NO:1-3, or an immunogenic fragment thereof, under conditions to elicit anantibody response, b) isolating antibodies from said animal, and c)screening the isolated antibodies with the polypeptide, therebyidentifying a polyclonal antibody which binds specifically to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-3.
 36. An antibody produced by a method ofclaim
 35. 37. A composition comprising the antibody of claim 36 and asuitable carrier.
 38. A method of making a monoclonal antibody with thespecificity of the antibody of claim 10, the method comprising: a)immunizing an animal with a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-3, or an immunogenicfragment thereof, under conditions to elicit an antibody response, b)isolating antibody producing cells from the animal, c) fusing theantibody producing cells with immortalized cells to form monoclonalantibody- producing hybridoma cells, d) culturing the hybridoma cells,and e) isolating from the culture monoclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1-3.
 39. A monoclonal antibodyproduced by a method of claim
 38. 40. A composition comprising theantibody of claim 39 and a suitable carrier.
 41. The antibody of claim10, wherein the antibody is produced by screening a Fab expressionlibrary.
 42. The antibody of claim 10, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 43. A method ofdetecting a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-3 in a sample, the method comprising:a) incubating the antibody of claim 10 with a sample under conditions toallow specific binding of the antibody and the polypeptide, and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-3 in the sample.
 44. A method ofpurifying a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-3 from a sample, the method comprising:a) incubating the antibody of claim 10 with a sample under conditions toallow specific binding of the antibody and the polypeptide, and b)separating the antibody from the sample and obtaining the purifiedpolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-3.
 45. A polypeptide of claim 1, comprisingthe amino acid sequence of SEQ ID NO: 1
 46. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO :
 2. 46. A polypeptideof claim 1, comprising the amino acid sequence of SEQ ID NO:
 3. 47. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO : 4
 48. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:
 5. 49. A polynucleotide of claim11, comprising the polynucleotide sequence of SEQ ID NO: 6.